Angiogenesis, the process of new blood vessel development and formation, plays an important role in numerous physiological events, both normal and pathological. Angiogenesis occurs in response to specific signals and involves a complex process characterized by infiltration of the basal lamina by vascular endothelial cells in response to angiogenic growth signal(s), migration of the endothelial cells toward the source of the signal(s), and subsequent proliferation and formation of the capillary tube. Blood flow through the newly formed capillary is initiated after the endothelial cells come into contact and connect with a preexisting capillary.
Angiogenesis is indispensable for embryonic development, organogenesis, tissue regeneration and repair, wound healing and female reproductive processes (Folkman, J. And Shing, Y., J. Biol. Chem. 267:109931–10934, 1992; Folkman, J., Nature Medicine 1: 27–31, 1995). Meanwhile, angiogenesis is also one of the necessary factors that cause the progression and deterioration of many pathological disorders including cancer growth and metastasis, cardiovascular disease, diabetic retinopathy, rheumatoid arthritis, etc. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis.
Angiogenesis is a complex multi-stage process that includes proliferation, migration and differentiation of endothelial cells, proteolytic degradation of the basement membrane, differentiation and migration of endothelial cells into the surrounding stroma, and finally formation of vasculature and new capillaries. The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate (Rastinejad et al., 1989, Cell 56:345–355).
Angiogenesis stimulators that can be mentioned include vascular endothelial growth factor (VEGF), vascular permeability factor (VPF), fibroblast growth factor (FGF-1 and -2), etc. On the other hand, some angiogenesis inhibitors have also been found and identified recently, which includes a 29 KDa fragment of fibronectin, thrombospondin (TSP-1), platelet factor 4, a 16 KDa fragment of prolactin, and a 38 KDa fragment of plasminogen and the like. In particular, recently O'Reilly et al. identified and characterized an internal 38 KDa fragment of plasminogen as angiostatin and a 20 KDa globular C-terminal of collagen XVIII as endostatin. It is suggested based on the current research results that the angiogenesis phenotype in the tissue depends on the dynamic equilibrium of angiogenesis stimulator and inhibitors in the local tissue environment (Folkman, J., N. Engl. J. Med. 333: 1757–1763, 1995).
Particularly interesting is that recent research shows that most angiogenesis inhibitors as mentioned above display the inhibitory activity of endothelial cell proliferation only after their parent proteins are hydrolized and form terminal or internal fragments. Thus, it is suggested that protein hydrolysis by endogenous peptidases plays a key role in the expression of their biological activities (O'Reilly, M. S. et al., Cell 88:277–285, 1997).
As a 20 KDa carboxyl terminal fragment of collagen XVIII, endostatin is a special inhibitor of endothelial cell proliferation and migration, and it also markedly inhibits the growth of many kinds of cancers (O'Reilly, M. S. et al., Cell 88:277–285, 1997; U.S. Pat. No. 5,854,205). It was shown that repeated endostatin administration leads to prolonged stable state of mice cancers, and there was no induction of drug resistance (Boehm, T. et al., Nature 390:404–407, 1997). It was further shown that endostatin causes cells to be quiescent at cell cycle G1 phase and specifically induces apoptosis of endothelial cells (Dhanabalk, M. et al., Biochem. Biophys. Res. Commun. 258: 345–352, 1999).
Endostatin was initially isolated from a hemangioendothelioma cell line for its ability to inhibit the proliferation of capillary endothelial cells (O'Reilly, M. S. et al., Cell 88:277–285, 1997). Based on the analysis of its nucleotide sequence, O'Reilly et al. further expressed endostatin protein in an E. coli expression system in un-refolded form, and it is believed that the unfolded purified protein facilitates its prolonged release at the subcutaneous injection site. The authors also mentioned that when endostatin was refolded by a standard method and solublized into tissue culture media, it strongly inhibited the proliferation of endothelial cells. Unfortunately, about 99% of protein was lost during protein refolding. In addition, though it has been reported that protein having anti-angiogenesis activity can be expressed in prokaryotes, the product can hardly refold into soluble form and tends to precipitate out of the solution. Further, cloning and expressing soluble recombinant endostatin in a yeast (Pichia pastoris) system were also reported (see, for example, Dhanabal, M. Et al., Cancer Res. 59: 189–197, 1999).
Yuan-Hua Ding and his co-workers (Yuan-Hua Ding et al., Proc. Natl. Acad. Sci. USA 95,10443–10448, 1998) revealed a zinc-binding site in the structure analysis of human endostatin by x-ray crystallography, and supposed that the zinc site could be involved either in the cleavage of the precursor or in some activity following cleavage. The writers concluded that “strategies for stabilizing the zinc-binding loop with other metals may be useful to stabilize the protein, especially if the loop contacts a receptor. Protein engineering to create a convalent dimer by a disulfide link or by forming a single chain dimer based on the proximity of the C and N termini of respective monomers, might produce a more potent protein.” Also, the previous study by Boehm et al. (Boehm, T. et al., Biochem. Biophys. Res. Commun., 252:190–194,1998 ) suggested that zinc-binding is essential for the angiogenesis activity of endostatin.
However, contrary to the conclusion of the studies discussed above, B. K. L. Sim and his co-workers (B. Kim Lee Sim et al., Angiogenesis 3,41–51,1999 ) have shown that deletion of two (His1 and His3) of the four zinc ligands of recombinant human (rh) Endostatin did not affect the inhibitory activity of the protein in vivo. However, for these completely different results, the writers did not give a satisfactory explanation.
Furthermore, Sim et al. demonstrated soluble and unrefolded rhEndostatin produced in P. pastoris exhibits an inhibition of 40–98% in the B16-BL6 metastatic tumor model, but the effective inhibitory dose up to 50 mg/kg/12 h. Additionally, it has been shown that (2001, 2002 ASCO Annual Meeting Report: www.entremed.com) the effective amount of endostatin expressed in yeast system is astonishingly about 240–600 mg/m2/person in recent clinical trials. Obviously, such a high dose must have a great stress on large scale manufacture for clinical trials and on industrial production in the future, even though it appears safe in high doses as determined in mouse and monkeys. Also, to reduce the dosage and to increase the effect, continuous infusion with pumps has been tried in their clinical trials. However, this will make the patients suffer a great inconvenience, and it does not lead to an obviously improved effect.
Because of the huge dosage of endostatin used as cancer growth inhibitor in the preclinical and clinical studies, in addition to the problem of being difficult to refold and easy to precipitate for product expressed in E. coli, secreted recombinant human endostatin that is expressed in a yeast expression system is currently preferred. But use of the yeast system demands huge amount of investment (for example up to 10 or more tons of fermentor), long production cycles, and suffers the danger of huge losses incurred by possible contamination.
Due to the problems existing in the practical application for recombinant human endostatin, it is necessary to seek a new production method and product that has higher yield, low cost, and better in vivo stability in order to improve the clinical effects of recombinant human endostatin in the treatment of cancers or other angiogenesis related diseases.
China Patent No. 00107569.1 (its publication date is Dec. 5, 2001) described endostatin with elongated N-terminal or additional amino acids at the N-terminal, and a method for production in prokaryotes (E. coli, for example ) expression system. The human endostatin with additional amino acids at the N-terminal is produced in refolded form in high yield, thereby supporting large scale manufacture for clinical evaluation. Recently, on the basis of our previous experimental research, our further pharmacological and pharmacokinetics studies showed that human endostatin with N-terminal additional amino acid sequence as disclosed in China Patent No. 00107569.1 has better in vivo stability and biological activity than our previously produced native endostatin in yeast, thereby markedly improving its pharmaceutical activity and substantially decreasing the clinical administration doses. These findings and improvements of the present inventors provide the desirability and availability of the modified rhEndostatin protein for the use in clinical practices.